Secondary metabolites display a broad range of useful antibiotic and immunosuppressant activities as well as less desirable phyto- and mycotoxic activities. (Demain, A., and Fang, A. (2000) The Natural Functions of Secondary Metabolites. In Advances in Biochemical Engineering/Biotechnology, T. Scheper, ed. (Berlin Heidelberg, Springer-Verlag), pp. 1-39). For example, penicillin and derivatives, produced by Aspergillus, Cephalosporium and Penicillium species are widely used antibiotics (Brakhage, A. A. (1998) Microbiol. Mol. Biol. Rev. 62, 547-585), lovastatin is a potent cholesterol lowering drug produced by Aspergillus terreus (Kennedy et al. (1999) Science 284, 1368-1372) and aflatoxins, produced by several Aspergillus species, are highly toxic carcinogens contaminating many crops (Hicks et al. (2002) Genetics and Biosynthesis of Aflatoxins and Sterigmatocystin. in The Mycota, Vol. XI, Kempken and Bennett, eds. (Spring-Verlag). The distribution of natural products is characteristically restricted to certain fungal taxa, particularly the Ascomycetes. Perhaps the greatest number of known secondary metabolites has been ascribed to the Ascomycete genus Emericella (asexual stage=Aspergillus). (reviewed in Brakhage, 1998 and Hicks et al., 2002 respectively).
Much of the current understanding of fungal secondary metabolite regulation arises from studies of the genetic model Aspergillus nidulans. This organism produces many natural products including sterigmatocystin ST (ST; the penultimate precursor to aflatoxin) (Hicks et al., 2002) and penicillin (Brakhage, 1998; Penalva et al. (1998) Trends Biotechnol. 16, 483-489) and has been used as a heterologous host to study the biosynthesis of other natural products including lovastatin (Kennedy et al., 1999). Critical advances in understanding of fungal secondary metabolism have been largely based on primary studies from A. nidulans and/or secondary studies in other fungi where researchers were able to exploit the knowledge gained from A. nidulans to their fungus of choice (Tag et al. (2000) Mol. Microbiol. 38, 658-665; Borgia et al. (1994) FEMS Microbiol. Lett. 122, 227-231; Shen et al. (1998) Genetics 148, 1031-1041). These advances include the discovery of a penicillin (Montenegro et al. (1992) J. Bacteriol. 174, 7063-7067) and ST biosynthetic gene cluster (Brown et al. (1996) Proc. Natl. Acad. Sci. USA 93, 1418-1422) and the establishment of a G-protein/cAMP/protein kinase A mediated growth pathway in A. nidulans regulating secondary metabolism production and sporulation (Hicks et al., 1997; Tag et al., 2000; Shimizu et al. (2001) Genetics 157, 591-600).
Through the use of Aspergillus nidulans, it is now apparent that structural genes required for most secondary metabolites are clustered (Keller et al. (1997) Fungal Genetics and Biology 21, 17-29), that the regulation of the clustered genes is largely dependent on pathway specific transcription factors (Fernandes et al. (1998) Mo. Microbiol 28, 1355-1365; Hohn et al (1999) Fungal Genet. Biol. 26, 224-235; Tsuji et al. (2000) Mol. Microbiol. 38, 940-954) and that G protein regulation of fungal secondary metabolism is likely to be a conserved phenomena (Tag et al., 2000) presumably transmitted through the pathway specific transcription factor.
The biosynthetic genes necessary for sterigmatocystin (ST) production in A. nidulans are clustered on a ca. 60-kb region on chromosome IV (Brown et al., 1996). The expression of these cluster genes (called stc genes) is regulated by the sixth gene in the cluster, aflR. aflR encodes a zinc binuclear cluster DNA binding protein which binds to AflR sites in stc promoters (Fernandes et al., 1998). ST is the penultimate precursor of aflatoxin (AF), which is produced by the related species A. flavus and A. parasitieus. AflR was first identified in A. favus (Payne et al. (1993) Appl. Environ Microbiol. 59, 156-162) and subsequently in A. parasiticus (Chang et al. (1993) Appl. Environ. Microbiol. 59, 3273-3279). AflR regulates the expression of the AF cluster genes in both A. flavus and A. parasiticus in a manner similar to the stc genes. aflR is not constitutively expressed in these three species and is regulated through a complex interaction with G protein/cAMP/protein kinase A signal transduction pathway also involved in asexual spore development (Hicks et al., 1997; Shimizu and Keller, 2001).
The discovery of G protein/cAMP/protein kinase A regulation of ST and other fungal secondary metabolites (Shimizu and Keller; 2001; Hicks et al., 2002; Tag et al., 2000) has been decidedly helpful in establishing a concept of global regulation of secondary metabolism. However, currently available signal transduction mutants have pleiotrophic effects on the fungi, the most notable effect being the gross impact on spore production and vegetative hyphal growth (Hicks et al., 1997; Tag et al., 2000; Shimizu and Keller, 2001; Adams et al. (1998) Curr. Opin. Microbiol. 1, 674-677). Thus, currently available signal transduction mutants are so impaired as to fungal development that further elucidation of genes specific for regulation of secondary metabolite gene clusters is difficult.
Studies of bacteria have only recently identified unique proteins whose primary function appears to be directed to regulation of multiple groups of secondary metabolism genes. These include AfsR, a transcriptional factor with ATPase activity regulating the production of actinorhodin, undecylprodigiosin and calcium-dependent antibiotic in Streptomyces coelicolor (Lee et al. (2002) Mol. Microbiol. 43, 1413-1430) and RsmA, a post transcriptional regulator of secondary metabolites and virulence factors in Pseudomonas aeruginosa (Pessi et al. (2001) J. Bacteriol. 183, 6676-6683). Similar proteins have not yet been identified in fungi but the existence of such in bacteria suggests the exciting possibility of global regulators of secondary metabolism in the Fungal Kingdom.
Although various similarities have been observed between secondary metabolite gene clusters in terms of cluster-specific regulatory elements, identification of regulatory elements providing global regulation of secondary metabolite gene clusters with little effect on sporulation and vegetative growth have not been reported. Such regulatory elements are extremely desirable because they would possess broad specificity for the activation and/or repression of entire families of secondary metabolite gene clusters while providing strains capable of otherwise normal or near-normal development and growth. Furthermore, identification of such regulatory elements would enable the increased production of secondary metabolites by providing improved strains of engineered organisms and also contribute to the broader understanding of molecular mechanisms by which secondary metabolites are produced.